Glioblastoma multiforme (GBM), or grade IV malignant glioma is an almost uniformly fatal brain tumor despite aggressive combination approaches using surgery, radiotherapy and chemotherapy. While radiation therapy has proven to be the most effective adjunctive modality, modifications, including altered fractionation, dose escalation with radiosurgery, conformal radiotherapy and brachytherapy, have not significantly improved the natural history of progression of these tumors. The thrust of this Program Project continues to focus on development of genetically engineered herpes simplex viruses (HSV) for improved therapy of these tumors. In the first 4 1/2 years of funding, we made the seminal observation that radiation synergistically improved regressions and cure-rates of heterotopic and orthotopic human GBM xenografts treated with genetically engineered HSV-1. We showed that modest doses of radiation enhanced proliferation and spread of genetically altered HSV-1 throughout the tumor bed in flank and intracranial GBM xenografts. Thus, we hypothesize that the biology of HSV-1 and the molecular response of GBM to ionizing radiation can be integrated to provide a basis for the translation of HSV-1 and therapeutic radiation to the clinical setting. Therefore, we propose to (i) identify doses and timing of radiation to maximize HSV-1 replication in GBM, (ii) identify genes that mediate radioresistancc in GBM and test whether these genes mediate HSV-1 proliferation and spread following ionizing radiation, (iii) validate genes in Aim 2 or identify other cellular genes that mediate HSV-1 proliferation and spread following radiotherapy, and (iv) select viruses that have an increased ability to replicate in radioresistant cells or under conditions of radiation. Our studies will rationally develop new strategies and reagents to significantly improve treatment of GBM.